The extracellular matrix is a complex web of proteins and polysaccharides that provides the mechanical scaffold that guides the development and preserves the integrity of organs and tissues. The proteins of the extracellular matrix are exposed to a broad range of mechanical forces that can reach into the hundreds of pN per molecule. However, our understanding of protein dynamics under force remains limited because the nanomechanics of proteins cannot be studied using solution biochemistry. Single molecule force-spectroscopy studies, at the nanometer scale, have demonstrated that in response to a stretching force, proteins undergo unfolding/refolding cycles providing for an elastic response while uncovering cryptic binding sites involved in mechanical signal transduction. Force also has other consequences such as altering the rate of chemical reactions. While the existence of these phenomena is now well documented, a molecular level understanding of the dynamics of proteins under force is still lacking. During the past funding period of this grant we developed the force-clamp spectroscopy technique. This technique directly measures the force-dependency of mechanical reactions in proteins. The force-dependency of a mechanical reaction can be directly related to the transition state structure, which determines the rate of the reaction. The mechanical transition state determines how a protein will behave when exposed to a stretching force. Here we propose to use these novel approaches to measure the force dependency of several important mechanical reactions such as folding, unfolding and the reduction of disulfide bonds by small nucleophiles. These experiments will examine the mechanical unfolding transition states of crucial proteins involved in the elasticity and signaling of the extracellular matrix: fibronectin, and talin and model proteins such as ubiquitin and the I27 immunoglobulin protein. Upon a reduction in the pulling force, these proteins rapidly fold, turning off their mechano-chemical signals. However, very little is known of how proteins fold under force. Here we will use a variety of force-clamp protocols to identify and characterize the individual stages visited by these proteins during their individual mechanical folding trajectories. We will also study another critical process governing the elasticity and folding of proteins of the extracellular matrix; disulfide bond reduction under force. We will use our sensitive force-clamp assay to examine how a mechanical stretching force applied to a protein alters the chemical mechanisms of disulfide bond reduction. The proposed experiments will uncover the molecular details of the structures that dominate the dynamics of proteins exposed to mechanical forces, crucial to understanding protein elasticity and mechano-chemical signaling in the extracellular matrix. PUBLIC HEALTH RELEVANCE: The elasticity of healthy and diseased tissues is determined by how proteins respond to mechanical stress. It is then of great medical importance to understand the nanomechanical properties of proteins. Force-clamp spectroscopy now provides a detailed view of the key structures involved in the mechanical responses of proteins. These studies provide a fundamental understanding of the roles played by proteins in the assembly of elastic tissues in all organisms.